Neutrophil extracellular traps and phagocytosis in Pythium insidiosum

Neutrophils are innate immune cells that play crucial roles in response to extracellular pathogens, including bacteria and fungi. Pythium insidiosum (P insidiosum) is a fungus-like pathogen that causes "pythiosis" in mammals. This study investigated in vitro function of human neutrophils against P. insidiosum. We demonstrated the killing mechanism of neutrophils when incubated with P. insidiosum zoospores (infective stage), such as phagocytosis and neutrophil extracellular traps (NETs). Healthy human neutrophils significantly reduced six strains of live zoospores isolated from different sources compared to the condition without neutrophils (p < 0.001), observed by colony count and trypan blue staining. As our results showed the killing ability of neutrophils, we further investigated the neutrophil killing mechanism when incubating with zoospores. Our study found that only two strains of heat-killed zoospores significantly induced phagocytosis (p < 0.01). Co-culture of heat-killed zoospores and neutrophils demonstrated NET formation, which was detected by immunofluorescence staining using DAPI, anti-myeloperoxidase, and anti-neutrophil elastase and quantitated under the fluorescence microscope. In addition, the level of cell-free DNA released from neutrophils (as a marker of NET production) after incubation with zoospores showed significantly increased levels when compared with unstimulated neutrophils (p < 0.001). Our findings demonstrate that neutrophils revealed the NET formation in response to P. insidiosum zoospores. This study is the first observation of the neutrophil mechanism against P. insidiosum, which could provide a better understanding of some parts of the innate immune response during pythiosis.


Introduction
Pythium insidiosum (P. insidiosum) is a species of oomycetes fungus-like pathogen found in moist soil and swampy area [1,2]. This pathogen causes "pythiosis" in mammals (e.g., humans, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 cattle, dogs, and cats), which usually occurs in tropical and subtropical areas [3][4][5]. The clinical manifestations of human infections can occur in four forms; vascular, cutaneous/subcutaneous, ocular, and disseminated pythiosis [6]. An asexual reproductive stage of this organism develops motile zoospores in a wet environment associated with its habitats and the prevalence area of this disease [7]. The infection starts once those zoospores attach to the host. After infection, the innate immune system is the primary line of defense to fight against invading pathogens, divided into two groups; barriers and protein molecules that prevent the infection and innate immune cells that recognize the pathogens and activate the effector cells [8]. However, understanding innate immune cells against P. insidiosum to reveal their pathogenesis has been limited. Thus, investigating neutrophils as a part of the innate immune response to P. insidiosum infection may provide a better insight into pathogenesis.
Neutrophils are important innate immune cells and are the most abundant polymorphonuclear leukocytes in human circulation, which play crucial roles during infection and inflammation [9,10]. The main functions of neutrophils include phagocytosis, degranulation, reactive oxygen species (ROS) production, and neutrophil extracellular traps (NETs) formation [11,12]. Evidence has shown that neutrophils are the first cells that migrate to the site of infection and eliminate pathogens [13,14]. For fungal infection, the yeast form of Candida albicans (C. albicans) was phagocytosed by neutrophils, whereas pseudohyphae were entrapped by NETs [15]. Moreover, the hyphal form of C. albicans can induce ROS production generated by the NADPH oxidase system [16]. Aspergillus fumigatus (A. fumigatus) hyphae were killed by toxic granules released by neutrophils [17]. Phialophora verrucosa (P. verrucosa) conidia induced NET formation [18]. These data support that neutrophils play vital roles in response to several pathogens.
The immunological study of pythiosis based on the equine model showed that infectious zoospores and geminated hyphae activated host immune response, including T helper 2 (Th2) response, and white blood cell migration (e.g., eosinophil and basophil), which revealed the inflammation and tissue damage [19]. In contrast, infected animals who received P. insidiosum antigen (PIA) as immunotherapy demonstrated the Th1 response, stimulating cell-mediated cytotoxicity, which facilitated microbe killing [19,20]. These findings were consistent with our previously unpublished data that showed increased levels of IFN-γ, IL-10, and IL-17 in Thai vascular pythiosis patients after receiving PIA immunotherapy. Noticeably, IL-17 activates neutrophil accumulation at the infection site [10,21], suggesting that neutrophils are involved in the pathogenesis of pythiosis.
Only a few studies have demonstrated neutrophils' role during pythiosis infection. This study investigated the functions of human neutrophils after co-culture with P. insidiosum, which are killing ability, phagocytosis, and NET formation.

Blood collection and neutrophil isolation
Heparinized blood from healthy donors (n = 6) was collected by phlebotomy with written informed consent. This study was approved by the Institutional Review Board (IRB) of the Faculty of Medicine, Chulalongkorn University, Thailand (COA No: 572/2020). Fresh heparinized blood was layered on Polymorphprep TM (Axis-shield, Norway) and centrifuged at 1,800 rpm for 30 min, 25˚C for neutrophil isolation, according to the manufacturer's instructions. The isolated neutrophils were collected, and cells were resuspended in RPMI 1640 medium with 25 mM HEPES and 2 mM L-glutamine (Gibco, USA), supplemented with 10% (v/v) fetal bovine serum (Gibco, USA). The contaminated red blood cells were lysed by 1x red cell lysis buffer (BioLegend, San Diego, CA). Cell viability and purity were determined by trypan blue and Wright-Giemsa staining, respectively.

Microorganisms and zoospore production
Six strains of P. insidiosum from different sources were selected; ATCC 58643 (CBS 574.85) from an equine, CBS 101039 from an Indian patient, CBS 777.85 from an equine, PC10 from a Thai patient, ATCC 90586 from an American patient, and PEC1 from water environment in Pasak Chonlasith Dam, Thailand. These isolates were grown in Sabouraud dextrose broth (SDB, Oxoid, UK). The zoosporogenesis technique was modified from Mendoza and Prendas [22]. The pathogens were cultured on sterile grass leaves (Axonopus compressus). After incubating at 37˚C for 48 h, grass leaves with hyphae were further transferred into the induction medium to generate encysted zoospores. The induction medium contains calcium and magnesium ions that facilitate the development of zoospores by all Pythium species [5]. The encysted zoospores were harvested from the induction medium and washed with 1x phosphate-buffered saline (PBS, Biolegend, USA). Then, zoospore numbers were counted by a hemocytometer. All six strains of zoospores were individually used in all experiments; neutrophil killing assay, phagocytosis, and NET formation.

Killing assay
The killing assay protocol was slightly modified from Gazendam RP et al. [17]. Co-culture of 2 × 10 5 encysted zoospores opsonized with heat-inactivated pooled serum and 2 × 10 5 neutrophils was performed for 2 h at 37˚C in a 5% CO 2 incubator. After incubation, neutrophils were lysed in sterile distilled water for 5 min. Samples were centrifuged at 10˚C, 5,000 rpm for 10 min. Two assays observed the neutrophil-killing activity. The 0.4% trypan blue staining with a 1:4 ratio (zoospores:dye) was performed to show the nonviable zoospores under a 40x light microscope (Olympus BX50, Japan). Confirmation by colony count was done in addition to the staining. The samples were diluted with 1X PBS and then cultured on blood agar (Oxoid, UK), a recommended media for P. insidiosum culture [7]. After the incubation at 37˚C for 15 h, colonies were counted and compared with the condition without neutrophils. All experiments were processed in triplicate independently.

Phagocytosis
Neutrophil phagocytic activity was measured using pHrodo-succinimidyl ester (red fluorescence) (Invitrogen, USA). The staining protocols were slightly modified from Shintaku et al. [23]. Briefly, all zoospore isolates were heat-killed at 80˚C for 30 min with a shaker, and the heat-killed zoospores were centrifuged for 10 min at 5,000 rpm. The pellets were resuspended in 663 μg/ml pHrodo in 1x PBS, incubated for 30 min with a shaker, and protected from light. Stained zoospores were washed three times with 1x PBS.
The pHrodo labeled zoospores were incubated with isolated neutrophils in a 96-U bottom well cell culture plate (SPL Life Sciences, Korea) with a ratio of 1:1 after opsonization with 5% (v/v) heat-inactivated pooled serum. The samples of each strain were treated with 10 μg/ml of cytochalasin D (Sigma-Aldrich, Israel) as a negative control because cytochalasin D inhibits actin polymerization and blocks cell movement resulting in phagocytosis inhibition [24]. After incubation for 30 min at 37˚C in a 5% CO 2 incubator, the samples were blocked with flow cytometry staining buffer (or FACS buffer) supplemented with human AB serum at 4˚C for 20 min. The cells were stained with 0.2 mg/ml of anti-CD11b antibody (APC) as a neutrophil marker (Biolegend, USA) and washed with FACS buffer. Cells were stored on ice and then measured by a flow cytometer (FACsAria II, BD Biosciences, USA). Phagocytosed zoospores-pHrodo inside neutrophils were detected in the PE channel at 575 nm wavelength, which indicated PE-positive cells.

NET formation
An immunofluorescence assay was used for NET formation from healthy human neutrophils induced with P. insidiosum zoospores. Sterile glass coverslips were placed in a 24-well flatshaped bottom plate (Jet Bio-Filtration, China) and coated with poly-L-lysine (Sigma-Aldrich, Germany) for neutrophil attachment. After washing with 1xPBS, neutrophils were seeded and incubated for 1 h at 37˚C in a 5% CO 2 incubator. The heat-killed zoospores or 100 ng/ml phorbol myristate acetate (PMA, as a positive control, Sigma-Aldrich, USA) were added and incubated for 2 h. After incubation, the culture medium was gently aspirated for DNA quantification. The glass coverslips were fixed and permeabilized with 1% formaldehyde and 1X Tris-buffered saline (TBS) in 0.05% tween 20, respectively. The samples were blocked with 1xTBS with 2% bovine serum albumin (BSA) for 30 min. The staining protocol was slightly modified from Sae-khow et al. [25]. Samples were stained with 4',6-Diamidino-2-phenylindole (DAPI, Thermo fisher scientific, USA), rabbit anti-neutrophil elastase, and mouse anti-myeloperoxidase (Abcam, UK) for DNA, elastase, and myeloperoxidase detection, respectively. Primary antibodies were detected with goat anti-rabbit IgG Alexa fluor 488 and goat anti-mouse IgG Alexa fluor 647 (Abcam, UK) under a fluorescence microscope (Olympus IX81, Japan) and confocal laser scanning microscope (ZEISS LSM800, Germany). The percentage of NET formation was calculated by counting the number of NET-forming cells per 100 neutrophils, which was processed in triplicate independently.

Double-stranded DNA (dsDNA) quantification
After co-incubation for 2 h, the culture medium described in NET formation was prepared for dsDNA determination. 0.1 M CaCl 2 and 50 U/ml micrococcal nuclease (Sigma-Aldrich, USA) were added for 10 min for DNA fragmentation and digestion. Next, 0.5 M ethylenediaminetetraacetic acid (EDTA) was added to stop the reaction. Then, the supernatants were collected and incubated with Quant-iT TM PicoGreen1 reagent (Invitrogen, UK) in a ratio of 1:1 to determine the cell-free DNA, according to the manufacturer's instructions. After incubation for 5 min in the dark, the DNA mixture was measured at an excitation wavelength of 480 nm and emission wavelength of 530 nm by spectrofluorometer (Varioskan Flash, Thermo fisher scientific, Finland).

Statistical analyses
Statistical analyses were performed by GraphPad version 5.03 (GraphPad Software, San Diego, CA). Data are presented as mean ± standard error of the mean (SEM). One-way ANOVA followed by Bonferroni analysis was used to compare the groups. Differences with a p-value of � p < 0.05, �� p < 0.01, and p < 0.001 were considered statistically significant.

P. insidiosum phagocytosed by human neutrophils
As neutrophils reduced colony counts and zoospore viability of P. insidiosum, we investigated whether neutrophils eliminate this pathogen through phagocytosis. Healthy neutrophils were incubated with opsonized heat-killed pHrodo labeled P. insidiosum zoospores. Phagocytosed zoospores-PE inside neutrophils (PE-positive cells) were gated and detected using flow cytometry. Neutrophils pretreated with cytochalasin D were used as a negative control (Fig 2A and  2B). The results showed that four strains of zoospores, PEC1, CBS 777.85, CBS101039, and ATCC 58643, were phagocytosed by neutrophils. However, the percentages of phagocytosis were significantly increased only in zoospores from ATCC 58643 and CBS 777.85 strains; 3.6 ± 1.2% (p < 0.01) and 3.2 ± 0.9% (p < 0.05, n = 6), respectively when compared with the negative control (Fig 2C).

Discussion
Pythiosis is a life-threatening disease caused by P. insidiosum, the fungus-like microbe [1,4,7]. In the initial phase of infection, the innate immune system acts as the primary line of defense to fight against invading pathogens [8]. Understanding the human immune response to P. insidiosum, especially innate immunity, is still unclear [26][27][28]. Neutrophils are one of the important granulocytic innate immune cells that play essential roles in response against pathogens [13]. However, no report demonstrates neutrophils' response against P. insidiosum. This study, therefore, investigated the killing ability of neutrophils and two primary neutrophil mechanisms, which were composed of phagocytosis and NET formation, in response to P. insidiosum.
In this study, we first explored the killing activity of human neutrophils against P. insidiosum. We determined whether human neutrophils decreased the colony number and viability of zoospores (infective stage) of P. insidiosum. As predicted, all different strain colony counts on blood agar were significantly reduced when neutrophils were added to the zoospores. The significant reduction of zoospore viability supported this result after these zoospores were cocultured with human neutrophils and stained with trypan blue to discriminate live/dead zoospores [29]. However, complete colony count reductions and zoospore viability were not observed. These findings indicate that neutrophils are one part that plays a role in the elimination of P. insidiosum. Nevertheless, our results suggest that P. insidiosum was perhaps hardly destroyed by human neutrophils.
Phagocytosis is one of the significant mechanisms of neutrophils in response to invading pathogens. This study, therefore, investigated the phagocytosis of neutrophils in P. insidiosum. There were only 2 strains that showed significantly increased neutrophil phagocytosis. These findings suggest that phagocytosis is probably not the primary killing mechanism of P. insidiosum because the size of the zoospores of this pathogen is large, and neutrophils are probably unable to phagocytose zoospores of this pathogen easily. In addition, human neutrophils are 10-12 μm in diameter, while zoospores of P. insidiosum are 8-12 μm in diameter and are larger than the yeast form of C. albicans (2-3 μm in diameter) [30,31]. Branzk N et al. reported that the incubation of neutrophils and yeast-locked C. albicans induced neutrophil phagocytosis. In contrast, the incubation with the hyphal form of this C. albicans resulted in NET formation [15]. These data supported that phagocytosis of neutrophils depended on the microbial size and implied that neutrophils formed NETs when they encountered large-sized pathogens such as P. insidiosum.
As NETs are another essential mechanism of neutrophils in fungal infection, particularly large-sized pathogens (e.g., zoospores of P. insidiosum), this study determined NET formation after human neutrophils were incubated with P. insidiosum zoospores. Neutrophils released the DNA web-like structure containing elastase and MPO granules but were not observed in untreated neutrophils. In addition, the levels of NET products or cell-free DNA supported these findings. Neutrophils demonstrate the essential roles of NET in response to many fungi, such as Pialophora verrucosa [18], Aspergillus fumigatus [17], Curvularia lunata [32], etc., resulting in the production of NADPH oxidase, MPO and elastase releasing, and decreasing of fungal viability. Our observation supports that NET formation may be the primary killing mechanism of P. insidiosum.
In conclusion, this study demonstrated that human neutrophils kill P. insidiosum via NET formation and partly phagocytosis. As large-sized pathogens such as P. insidiosum zoospores are the infective stage causing pythiosis, this study supported the idea that NETs become a key player in eliminating these pathogens. However, these observations could not represent the exact pathogenesis of neutrophils during P. insidiosum infection because heat-killed zoospores were used in both phagocytosis and NET assays. Dead microorganisms show lower immune stimulation than live pathogenic microorganisms due to their virulence factors and some protein molecules [33]. Killed zoospores lack some activities (e.g., hyphal germination) and some secretory components (e.g., protease), which may decrease the ability of immune stimulation. Confirming phagocytosis and NET formation by human neutrophils incubating with live zoospores is needed. Moreover, future studies should increase the neutrophil population in the case of healthy donors and pythiosis patients and zoospores induced from different hosts to explore how neutrophils function. This information will shed light on new management and therapy in pythiosis patients.